Limits...
Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus

Gene structure and amino acid alignment of GmF3G2″Gt. (A) Intron-exon structure of GmF3G2″Gt gene. (B) Amino acid alignment of soybean GmF3G2″Gt-a, morning glory Ip3GGT encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase and Arabidopsis UGT79B6 that functions as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase. Identical amino acids are in white font highlighted in black, similar amino acids are in white font highlighted in gray. Four amino acids that differed in GmF3G2″Gt-b are exhibited below the aligned sequence in red font.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4494776&req=5

Fig4: Gene structure and amino acid alignment of GmF3G2″Gt. (A) Intron-exon structure of GmF3G2″Gt gene. (B) Amino acid alignment of soybean GmF3G2″Gt-a, morning glory Ip3GGT encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase and Arabidopsis UGT79B6 that functions as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase. Identical amino acids are in white font highlighted in black, similar amino acids are in white font highlighted in gray. Four amino acids that differed in GmF3G2″Gt-b are exhibited below the aligned sequence in red font.

Mentions: Survey of the genome sequence of a US cultivar Williams 82 suggested the existence of a gene similar to the GT gene, Glyma06g43880 between Satt307 and Sat_202. The entire coding region of Glyma06g43880 was amplified by PCR and cloned. Sequence analysis revealed that the open reading frame of Glyma06g43880 is 1380 bp long encoding 459 amino acids. We designated the gene as GmF3G2″Gt. GmF3G2" Gt belongs to the family 1 glycosyltransferase, and it was designated as UGT79B30 by the UGT Nomenclature Committee [25]. The flavonoid glycosyltransferase phylogenetic tree suggested that GmF3G2″Gt belongs to the flavonoid glycoside glycosyltransferase (GGT) gene cluster (Figure 3). BLAST analysis suggested that it had a 55% amino acid similarity with Ip3GGT of morning glory encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase [26] and 45% similarity with Arabidopsis UGT79B6 that was recently identified to function as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase [22] (Figure 3). Comparison with the genome sequence of Williams 82 suggested that GmF3G2″Gt had one intron (Figure 4A). Eight nucleotides were polymorphic between Harosoy and Nezumisaya; consisting of six single nucleotide polymorphisms (SNPs) and one two-nucleotide substitution. The nucleotide polymorphism resulted in four amino acid substitutions between the cultivars (amino acid positions, 20, 142, 149 and 183) (Figure 4B). The cDNA fragments generated from Nezumisaya and Harosoy were designated as GmF3G2″Gt-a and GmF3G2″Gt-b, respectively. The 5′ upstream region of about 1.8 kb was amplified by PCR from Harosoy, whereas the corresponding region could not be amplified from Nezumisaya. So we cloned the 5′ upstream region of both cultivars by genome walking. The nucleotide sequences of the 5′ upstream region, exons and introns of Harosoy were identical with those of Williams 82. In contrast, Nezumisaya had many indels and substitutions in the 5′ upstream region compared with Harosoy (Additional file 1: Figure S1).Figure 3


Linkage mapping, molecular cloning and functional analysis of soybean gene Fg3 encoding flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase.

Di S, Yan F, Rodas FR, Rodriguez TO, Murai Y, Iwashina T, Sugawara S, Mori T, Nakabayashi R, Yonekura-Sakakibara K, Saito K, Takahashi R - BMC Plant Biol. (2015)

Gene structure and amino acid alignment of GmF3G2″Gt. (A) Intron-exon structure of GmF3G2″Gt gene. (B) Amino acid alignment of soybean GmF3G2″Gt-a, morning glory Ip3GGT encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase and Arabidopsis UGT79B6 that functions as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase. Identical amino acids are in white font highlighted in black, similar amino acids are in white font highlighted in gray. Four amino acids that differed in GmF3G2″Gt-b are exhibited below the aligned sequence in red font.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4494776&req=5

Fig4: Gene structure and amino acid alignment of GmF3G2″Gt. (A) Intron-exon structure of GmF3G2″Gt gene. (B) Amino acid alignment of soybean GmF3G2″Gt-a, morning glory Ip3GGT encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase and Arabidopsis UGT79B6 that functions as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase. Identical amino acids are in white font highlighted in black, similar amino acids are in white font highlighted in gray. Four amino acids that differed in GmF3G2″Gt-b are exhibited below the aligned sequence in red font.
Mentions: Survey of the genome sequence of a US cultivar Williams 82 suggested the existence of a gene similar to the GT gene, Glyma06g43880 between Satt307 and Sat_202. The entire coding region of Glyma06g43880 was amplified by PCR and cloned. Sequence analysis revealed that the open reading frame of Glyma06g43880 is 1380 bp long encoding 459 amino acids. We designated the gene as GmF3G2″Gt. GmF3G2" Gt belongs to the family 1 glycosyltransferase, and it was designated as UGT79B30 by the UGT Nomenclature Committee [25]. The flavonoid glycosyltransferase phylogenetic tree suggested that GmF3G2″Gt belongs to the flavonoid glycoside glycosyltransferase (GGT) gene cluster (Figure 3). BLAST analysis suggested that it had a 55% amino acid similarity with Ip3GGT of morning glory encoding anthocyanin 3-O-glucoside (1 → 2) glucosyltransferase [26] and 45% similarity with Arabidopsis UGT79B6 that was recently identified to function as a flavonol 3-O-glucoside (1 → 2) glucosyltransferase [22] (Figure 3). Comparison with the genome sequence of Williams 82 suggested that GmF3G2″Gt had one intron (Figure 4A). Eight nucleotides were polymorphic between Harosoy and Nezumisaya; consisting of six single nucleotide polymorphisms (SNPs) and one two-nucleotide substitution. The nucleotide polymorphism resulted in four amino acid substitutions between the cultivars (amino acid positions, 20, 142, 149 and 183) (Figure 4B). The cDNA fragments generated from Nezumisaya and Harosoy were designated as GmF3G2″Gt-a and GmF3G2″Gt-b, respectively. The 5′ upstream region of about 1.8 kb was amplified by PCR from Harosoy, whereas the corresponding region could not be amplified from Nezumisaya. So we cloned the 5′ upstream region of both cultivars by genome walking. The nucleotide sequences of the 5′ upstream region, exons and introns of Harosoy were identical with those of Williams 82. In contrast, Nezumisaya had many indels and substitutions in the 5′ upstream region compared with Harosoy (Additional file 1: Figure S1).Figure 3

Bottom Line: GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee.Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8518, Japan. dishaokang@affrc.go.jp.

ABSTRACT

Background: Flavonol glycosides (FGs) are major components of soybean leaves and there are substantial differences in FG composition among genotypes. The first objective of this study was to identify genes responsible for FG biosynthesis and to locate them in the soybean genome. The second objective was to clone the candidate genes and to verify their function. Recombinant inbred lines (RILs) were developed from a cross between cultivars Nezumisaya and Harosoy.

Results: HPLC comparison with authentic samples suggested that FGs having glucose at the 2″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Nezumisaya, whereas FGs of Harosoy were devoid of 2″-glucose. Conversely, FGs having glucose at the 6″-position of glucose or galactose that is bound to the 3-position of kaempferol were present in Harosoy, whereas these FGs were absent in Nezumisaya. Genetic analysis suggested that two genes control the pattern of attachment of these sugar moieties in FGs. One of the genes may be responsible for attachment of glucose to the 2″-position, probably encoding for a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase. Nezumisaya may have a dominant whereas Harosoy may have a recessive allele of the gene. Based on SSR analysis, linkage mapping and genome database survey, we cloned a candidate gene designated as GmF3G2″Gt in the molecular linkage group C2 (chromosome 6). The open reading frame of GmF3G2″Gt is 1380 bp long encoding 459 amino acids with four amino acid substitutions among the cultivars. The GmF3G2″Gt recombinant protein converted kaempferol 3-O-glucoside to kaempferol 3-O-sophoroside. GmF3G2″Gt of Nezumisaya showed a broad activity for kaempferol/quercetin 3-O-glucoside/galactoside derivatives but it did not glucosylate kaempferol 3-O-rhamnosyl-(1 → 4)-[rhamnosyl-(1 → 6)-glucoside] and 3-O-rhamnosyl-(1 → 4)-[glucosyl-(1 → 6)-glucoside].

Conclusion: GmF3G2″Gt encodes a flavonol 3-O-glucoside/galactoside (1 → 2) glucosyltransferase and corresponds to the Fg3 gene. GmF3G2″Gt was designated as UGT79B30 by the UGT Nomenclature Committee. Based on substrate specificity of GmF3G2″Gt, 2″-glucosylation of flavonol 3-O-glycoside may be irreconcilable with 4″-glycosylation in soybean leaves.

No MeSH data available.


Related in: MedlinePlus